axor12: a novel human g protein-coupled receptor ... a novel human g protein-coupled receptor, named

Download AXOR12: a novel human G protein-coupled receptor ... A novel human G protein-coupled receptor, named

Post on 14-Apr-2020

0 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • 1

    AXOR12: a novel human G protein-coupled receptor, activated by the peptide KiSS-1.

    Alison I. Muir2, Larissa Chamberlain8, Nabil A. Elshourbagy6, David Michalovich5§, Darren J.

    Moore8, Amy Calamari6, Philip G. Szekeres2, Henry M. Sarau7, Jon K. Chambers2, Paul

    Murdock3, Klaudia Steplewski6, Usman Shabon6, Jane E. Miller2, Susan E. Middleton2, John G.

    Darker4, Christopher G. C. Larminie5, Shelagh Wilson2, Derk J. Bergsma6 Piers Emson8, Richard

    Faull9, Karen L. Philpott1, David C. Harrison1*

    Depts of Neurology1, Discovery Biology2, Biotechnology and Genetics3, Discovery Chemistry4,

    Bioinformatics5, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW,

    UK

    Depts of Biotechnology and Genetics6, Pulmonary Biology7 GlaxoSmithKline, 709 Swedeland

    Road, King of Prussia, PA, USA

    Neurobiology Programme, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK8

    Dept of Anatomy with Radiology, University of Auckland, New Zealand9

    §Current Address: Inpharmatica Ltd., 60 Charlotte St, London, W1T 2NU, UK

    *Corresponding author

    Telephone: +44 (0)1279 622728, Fax: +44 (0)1279 622371

    Email: David_C_Harrison@gsk.com

    Running Title: AXOR12, a novel receptor activated by KiSS-1

    Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

    JBC Papers in Press. Published on May 31, 2001 as Manuscript M102743200 by guest on A

    pril 16, 2020 http://w

    w w

    .jbc.org/ D

    ow nloaded from

    http://www.jbc.org/

  • 2

    Summary

    A novel human G protein-coupled receptor, named AXOR12, exhibiting 81% homology to the

    rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous

    expression of AXOR12 in mammalian cells permitted the identification of three surrogate

    agonist peptides, all with a common C-terminal amidated motif. High potency agonism,

    indicative of a cognate ligand, was evident from peptides derived from KiSS-1, a gene whose

    expression prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase

    chain reaction (RT-PCR) was used to study the expression of AXOR12 and KiSS-1 in a variety

    of tissues. Highest levels of expression of AXOR12 mRNA were observed in brain, pituitary

    gland and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and

    brain. A polyclonal antibody raised to the C-terminus of AXOR12 was generated and used to

    show localization of the receptor to neurons in the cerebellum, cerebral cortex and brainstem.

    The biological significance of these expression patterns and the nature of the putative cognate

    ligand for AXOR12 are discussed.

    by guest on A pril 16, 2020

    http://w w

    w .jbc.org/

    D ow

    nloaded from

    http://www.jbc.org/

  • 3

    Introduction

    The G protein-coupled receptors (GPCRs) form a large family of membrane bound proteins

    which share a unique structural feature comprising 7 transmembrane alpha helices. These

    molecules act as receptors for a diverse range of extracellular signalling molecules including

    small molecules (amino acids, biogenic amines), lipids, small bioactive peptides and large

    polypeptides (1). They have been successfully used as drug targets by the pharmaceutical

    industry for a number of years. Attention has focussed on a number of proteins which are known

    to be GPCRs through structural homology, but for which no ligand has been identified – so

    called orphan receptors. At the same time as the recent discovery of new GPCRs, there has been

    a renewed focus on discovering potential novel peptides which may act as endogenous ligands

    for these receptors.

    Here, we describe the cloning of a novel human orphan receptor, a class I GPCR with sequence

    similarity to receptors for the neuropeptide galanin. This receptor was given the name AXOR12,

    in accordance with its position in a series of receptors identified in our organization. AXOR12

    has a high degree of homology to the rat orphan receptor GPR54 (2) (81% amino acid identity)

    which suggests that these two receptors may be orthologs. In order to identify a ligand for

    AXOR12 we expressed this receptor in mammalian cells, and screened the transfected cells in a

    functional assay against a library rich in known and putative peptide transmitters. While there

    was no activity in response to galanin, we identified three peptides which acted as low potency

    agonists of AXOR12. These peptides were all derived from invertebrates and shared a C-

    terminal LRF- or LRW-amide motif.

    During the preparation of this manuscript, a search of patent literature revealed the existence of

    additional, high potency agonists with sequence similarities to the surrogate agonists identified

    by guest on A pril 16, 2020

    http://w w

    w .jbc.org/

    D ow

    nloaded from

    http://www.jbc.org/

  • 4

    from the screen. These peptides were derived from a precursor known as KiSS-1. The KiSS-1

    gene was originally identified as being upregulated in melanoma cells which have lost the

    potential to metastasize following microcell mediated transfer of human chromosome 6 (3).

    Subsequent studies have shown that exogenous expression of KiSS-1 in other tumor cell lines

    also prevents metastasis (4). The mechanism by which this occurs remains largely unknown,

    however KiSS-1 has structural features that suggest that it may be the precursor of a secreted

    peptide with an LRF-amide motif at the C-terminus. We synthesized the putative processed

    products of KiSS-1 and observed that they acted as high potency agonists of AXOR12.

    In order to gain insight into the physiological role of this receptor, we used quantitative RT-PCR

    to localize the mRNA expression of AXOR12 and of KiSS-1 in a range of human tissues. We

    observed high levels of AXOR12 expression in the brain. Further RT-PCR analysis of brain

    expression revealed a distinctive pattern of mRNA localisation which was further explored by

    immunohistochemistry using an antibody raised to the extreme C-terminal tail of the receptor. by guest on A pril 16, 2020

    http://w w

    w .jbc.org/

    D ow

    nloaded from

    http://www.jbc.org/

  • 5

    Experimental Procedures

    Receptor Cloning

    The human AXOR12 gene was initially identified within a genomic clone (AC005379) as five

    coding exons interrupted by 4 introns. The full-length cDNA was obtained by a modification of

    a previously described cDNA capture method (5). Briefly, 5µg of plasmid DNA from a human

    brain cDNA library was screened with a biotinylated oligonucleotide (5'-biotin with 18 atom

    spacer) corresponding to the 5' coding region (5'-GATGCGGACCGTGACCAACTTCTAC-3').

    Two additional 40-mers ( 5'-GGAACTCGCTGGTCATCTACGTCATCTGCCGCCACAAGCC-

    3' and 5'-ATCGCCAACCTGGCGGCCACGGACGTGACCTTCCTCCTGTG-3'),

    corresponding to the immediate 5' and 3' regions of the biotinylated probe were also used as

    blocking oligos. Bacterial colonies from the second round of selection were screened by PCR

    using AXOR12 specific primers. Five positive clones were identified and the entire inserts were

    sequenced on both strands using an ABI sequencer. Two of the sequenced clones were identical

    to each other and to the full length AXOR12 cDNA predicted from the genomic DNA sequence.

    Heterologous Receptor Expression and Functional Analysis in Mammalian Cells

    The AXOR12 cDNA was subcloned into the mammalian expression vector pCDN (6) as

    described previously (7). Transient transfections of HEK293 cells with AXOR12 alone or

    AXOR12 co-transfected with Gqi5 (a chimeric G protein α subunit consisting of Gαq with the C-

    terminal 5 amino acids substituted with the corresponding amino acids from Gαi2, which

    facilitates GPCR signalling through phospholipase C) were prepared for functional studies. A

    Ca2+ mobilization assay using the microtiter plate-based Ca2+ mobilization fluorometric imaging

    plate reader (FLIPR) was performed as described previously (8). HEK293 cells co-transfected

    by guest on A pril 16, 2020

    http://w w

    w .jbc.org/

    D ow

    nloaded from

    http://www.jbc.org/

  • 6

    with AXOR12 and Gqi5 were screened against a large library of over 1500 known and putative

    GPCR agonists, including all available mammalian neuropeptides, as described previously (9).

    Peptides in this library were tested at a final concentration of > 100 nM and other potential small

    molecule agonists at > 1 µM.

    To obtain mammalian cells stably expressing AXOR12, the cDNA was subcloned into the vector

    pCD (a derivative of pCDN lacking the gene for neomycin resistance) and co-transfected with

    pCDN Gqi5 (10) into Chinese hamster ovary cells using Lipofectamine PLUS (Life

    Technologies) at a DNA ratio of 10:1 (CHO AXOR12:Gqi5 cells). Forty eight hours later the

    cells were seeded into 96 well plates at 103 cells per well and selected with G418 (Life

    Technologies) (400 µg/ml) and in the absence of nucleosides. Doubly selected cells were

    screened by Northern blotting to confirm AXOR12 expression and positive clones we

Recommended

View more >